1. Field of the Invention
The present invention relates to a pharmaceutical composition comprising Clostridium botulinum neurotoxin and to a method of stabilizing the same. Particularly, the present invention is directed to a stabilized pharmaceutical composition including C. botulinum Type A and a cyclodextrin.
2. Description of Related Art
Botulinum neurotoxins are produced from anaerobic bacillus Clostridium botulinum. Seven related protein neurotoxins, known as serotypes A through G, are produced by different strains of the bacillus. Each of the seven serotypes of the botulinum neurotoxins is a large protein having a molecular weight of about 150 kDa, and a 100 kDa heavy chain of amino acid residues and a 50 kDa light chain of amino acid residues coupled by at least one disulfide linkage.
Clostridium botulinum neurotoxins, which cause the disease of botulism by blocking the release of the neurotransmitter, acetylcholine at the neuromuscular junction, are the most toxic proteins currently known to mankind. Food-borne botulism results from the consumption of improperly stored foods in which anaerobic C. botulinum grows and releases the toxin. In other forms of botulism, C. botulinum also produces the neurotoxin resulting in toxigenesis. For example, wound botulism results when the spores of C. botulinum are introduced into an open skin abrasion. The colonization of the wound is followed by the release of botulinum toxin. Similarly, infant botulism results from the consumption of C. botulinum spores followed by colonization in the intestine and toxigenesis.
Although the botulinum neurotoxins are known to be the most lethal natural toxin known to man, these lethal poisons have become utilized in the medical community as drugs with many indications. In this regard, the botulinum neurotoxins have been used to treat strabismus, and local injections of botulinum neurotoxin are now considered a safe and efficacious treatment for many neurological and non-neurological conditions. Recently, it has been observed that botulinum neurotoxin is useful as a treatment for diseases of the gastrointestinal tract. Botulinum neurotoxin is not only potent in blocking skeletal neuromuscular transmission, but also block cholinergic nerve endings in the autonomic nervous system. The capability to inhibit contraction of smooth muscles of the gastrointestinal tract was first suggested based on in vitro observations and later demonstrated in vivo, it has also been shown that botulinum neurotoxin does not block non adrenergic non cholinergic responses mediated by nitric oxide. This has further promoted the interest to use botulinum neurotoxin as a treatment for overactive smooth muscles and sphincters, such as the lower esophageal sphincter to treat esophageal achalasia, or the internal anal sphincter to treat anal fissure.
Commercially available pharmaceutical compositions comprising botulinum toxin are marketed under the trademarks including BOTOX® (Allergan, Inc. Irvine Calif.), Dysport® (Ipsen Ltd. Berkshire, U.K.) and Myobloc® (Elan Corp. Dublin Ireland). Typically, the pharmaceutical compositions are sold as vacuum-dried form that must be reconstituted with a diluent prior to actual usage. One major drawback to using the commercially available botulinum toxin preparations is the very short shelf life of the composition. In this regard, the actual usage of the pharmaceutical composition should be administered within about four hours after reconstitution because the botulinum toxin is very susceptible to denaturation due to surface denaturaion, heat, and alkaline conditions.
The susceptible denaturation of the botulinum neurotoxin reconsitution necessiates a need for methods of preserving the botulinum neurotoxin. It has been found that the Clostridium botulinum bacteria secretes the neurotoxin along with a group of neurotoxin associated proteins (“NAPs”). Research has shown that not only do the NAPs have a critical role in the toxico-infection of the botulinum neurotoxins, but the NAPs have an important role in the toxicity of the botulinum neurotoxin. In particular, it has been demonstrated the oral toxicity of the neurotoxin type A decreases by 43,000-fold upon removal of the NAPs. It has also been shown that the NAPs act to protect the neurotoxin against various environmental conditions including exposure to proteases, acidity, and heat. See, Kitamura, M., Sakaguchi, S., and Sakaguchi, G. (1969) Significance of the 12S toxin of Clostridium botulinum type E. Bacteriol. 98, 1173-1178; Sugii, S., Ohishi, I., and Sakagch, G., (1977) Botulogenic properties of vegetables with special reference to molecular size of the toxin in them, J. Food Safety 1, 53-65. Accordingly, it has been suggested that the NAPs interact with the neurotoxin to protect it from adverse environmental conditions. This protective role has led to the hypothesis that NAPs are important for the preservation of the structural integrity of the neurotoxin, as well as preservation of its activity.
Cyclodextrins are cyclic multiclyclopyranose unites connected by alpha-(1-4) linkages. The most widely known cyclodextrins are A, B, G-cyclodextrins, and their derivatives. The cyclic nature of the cyclodextrins, the hydrophobic properties of their cavities and the hydrophilic properties of their outer surfaces enable them to interact with other chemicals and produce inclusion compounds which are characterized by improved solubilities and stabilities. For example, U.S. Pat. No. 6,818,662 to Ito et al. discloses that solfobutyl ether B-cyclodextrin increases the solubility and light stability of N-(3-chloro-4-morpholin-4-yl)phenyl-N′-hydroxyimodoformadinie.
Although cyclodextrins are known to form inclusion complexes with drugs, there has been no disclosure or suggestion that a cyclodextrin can stabilize a botulinum neurotoxin. In fact, it has been thought that cyclic polymers including cyclodextrins can not be used to preserve or stabilize a botulinum neurotoxin. See, U.S. Patent Application Publication 2003/0118598 to Hunt, in which it has been taught that the botulinum neurotoxin can not utilize the cyclodextrin cavity because the cavity is much smaller in size than the neurotoxin. Accordingly, cyclodextrins cannot form inclusion complexes with botulinum neurotoxin.
There remains a need for an efficient and economic method and system for preparing a pharmaceutical composition comprising botulinum neurotoxin and a method for stabilizing and preserving the botulinum neurotoxin. The present invention satisfies these needs.